Effects Of Enzyme Loading Research Articles

In situ Transesterification of Crambe Seeds with Dimethyl Carbonate Using an Enzymatic Catalyst

In situ transesterification of crambe seeds oil was investigated to produce fatty acid methyl esters using dimethyl carbonate as acyl acceptor and lipase Novozyme 435® as the catalyst. First, the effects of solvent-to-seeds ratio, seed pretreatment, and extraction time were evaluated on the seed oil removal, comparing the results with conventional Soxhlet extraction. Then, the effects of enzyme loading (5 to 30 wt.% based on the oil mass) and reaction time (60 to 540 min) were evaluated on the simultaneous extraction and reaction. The highest oil extraction yield (ca. 26 wt.%) was achieved at the solvent-to-seeds ratio of 6 mL g-1 and 360 min. The oil obtained from seeds that received thermal pretreatment showed a higher concentration of minor compounds. The highest ester content (76.71 wt.%) was obtained with an enzyme loading of 20 wt.%, solvent-to-seeds ratio of 6 mL g-1, for 480 min. The reaction samples showed a predominance of erucate and oleate esters, with identification of the co-products formation (glycerol carbonate and glycerol dicarbonate), and ca. 5 wt.% of acylglycerols.

Regioselective enzymatic hydrolysis of blackcurrant seed oil in supercritical CO2

Continuous flow hydrolysis of blackcurrant seed oil dissolved in supercritical carbon dioxide (scCO2) was catalyzed by immobilized lipase Lipozyme TL IM. HPLC-MS and acid-base titration were used to determine composition of oil and hydrolysis products comprised of tri-, di- and monoglycerides, free fatty acids and glycerol. The oil contained 10 % linolenic and 2 % stearidonic fatty acids (omega-3), 51 % linoleic and 19 % gamma-linolenic fatty acids (omega-6), 12 % oleic acid, 5 % palmitic acid, and 1% stearic acid. Effects of enzyme load (17−500 mg), pressure (15−30 MPa), temperature (30−50 °C), and CO2 flow rate (0.2–4 g/min) on the conversion were evaluated. The reaction was reversible and the time to reach steady state was enzyme load-dependent and took up to 200 min. A slight drop of enzyme activity after four experimental runs and total reaction time 17 h was indicated by the decrease of free fatty acid content in the product from 0.72 % to 0.69 %. Due to the regioselectivity of lipase-catalyzed hydrolysis, the omega-3/omega-6 ratio was increased from 17:83 in oil to 23:77 in produced diglycerides.

Lipase‐mediated synthesis of neopentyl glycol diester using a combination of reduced and standard pressure

AbstractNeopentyl glycol (NPG) diester is a biodegradable lubricant used in the machinery industry as it is suitable for the low‐temperature environment. NPG diester was successfully synthesized with fatty acid and NPG using immobilized lipase. Immobilization was carried out with a liquid enzyme, Eversa® Transform 2.0 (from Thermomyces lanuginosus) and Lewatit VP OC 1600 as a carrier. The Eversa immobilized lipase prepared in this study was compared with commercial lipases, such as Novozym 435, Lipozyme RM IM, and Lipozyme TL IM. The Eversa immobilized lipase was the most effective biocatalyst for the synthesis of NPG diesters. The effects of enzyme loading and temperature on conversion were explored at a standard pressure of 101.3 kPa. And then, the effect of reduced pressure was investigated in the range of 1.3 to 40.0 kPa under optimum conditions obtained at standard pressure. The maximum conversion of ca. 63% was achieved at the optimal temperature of 50°C and the enzyme loading of 5% (based on the total weight of the substrate) at standard pressure after 10 h. However, the conversion to NPG diester increased markedly up to 97% after 10 h, even though reduced pressure as low as 26.7 kPa was applied. No significant differences in the conversion to NPG diester between the reactions at constant reduced pressure and the combined reaction of reduced pressure and standard pressure were observed. This pressure combination method employed in this study demonstrated a novel, energy‐saving strategy for the synthesis of NPG diester.

Effects of Enzyme Loading, Incubation Time and Incubation Temperature on Sawdust Hydrolysis by Locally Produced Bacterial Xylanase

The present study aims to investigate the enzymatic hydrolysis conditions of bacterial xylanase on alternative cheaper substrate which is hardwood sawdust (SD) in order to produce reducing sugars (xylose). The bacterial xylanase was produced and secreted from the Bacillus sp. The wood industry in Malaysia has become a major source of foreign exchange across the globe for developing the countries. Therefore, more wood residues (sawdust) are produced during the logging and processing of wood. Most of the sawdust will be disposed into the landfills. In actual, the sawdust can be utilised into more valuable products such as in producing reducing sugars. Thus, previous researches have studied on xylose production from wooden sawdust using commercial xylanases, but only few with the bacterial xylanase. Therefore, a study on the best conditions of enzymatic hydrolysis in producing xylose from sawdust using bacterial xylanase is essential. Prior to the enzymatic hydrolysis, the hardwood sawdust was pre-treated by autoclave at 121°C for 20 min in order to breakdown the lignin linkage and obtain the hemicellulosic xylan (delignification). The enzymatic hydrolysis conditions such as enzyme loading, incubation time and incubation temperature were experimented by One-Factor-At-Time (OFAT) method. Based on the experiment, the fifth cycle pre-treated autoclaved sawdust showed 5.5-fold higher than the untreated sawdust. The best enzymatic hydrolysis conditions for xylose production were enzyme loading of 1.4%, incubation time of 30 min, and incubation temperature of 56.9 °C. These conditions also succeeded in producing 2.5-fold higher xylose than the one without the enzymatic hydrolysis. 

The effect of ball milling on birch, pine, reed, walnut shell enzymatic hydrolysis recalcitrance and the structure of the isolated residual enzyme lignin

Methodologies for the high-yield recovery of lignin with retention of its native CO bonded structure is an essential prerequisite for many novel high-end lignin applications. Enzymatic residual lignin isolation is such a methodology that leaves the lignin untouched by using enzymatic desaccharification. Thus, a series of representative lignocellulose substrates (birch, pine, walnut shell and reed) were evaluated for effective native lignin isolation, with emphasis on the effect on the lignin structure and purity. The effect of enzyme loading and ball milling severity were studied by tracking residual saccharides and the structural integrity of the isolated lignin. Prolongation of ball milling time could achieve a higher carbohydrate removal and avoid the loading of extra enzyme. However, the application of two or more steps of enzymatic hydrolysis with higher enzyme loading and short ball milling time was shown as an alternative to long ball milling time to achieve similar carbohydrate removal and avoid extensive decrease of the lignin molecular weight (MW). This MW decrease was caused by breaking of some linkages, but not too a large enough extent to cause significant differences in the 2D HSQC NMR spectra. The recalcitrance towards increased enzyme hydrolysis activity by ball milling was different for the four representative biomasses and followed an order of walnut shell > reed ≈ pine > birch by comprehensive analysis the obtained data. Overall, the results showed a clear two-way synergy between enzymatic treatment and ball milling efficiency to isolate lignin with high yield, high native linkage content, purity and minimal MW reduction.

Organosolvent Extraction Coupled with Steam Explosion Pretreatment of Wild Sugarcane and Optimization of Cellulase Activity using Response Surface Methodology

The present study focuses on the effect of the organosolvent extraction with steam explosion pretreatment and optimization of cellulase activity using the response surface methodology for the production of bioethanol. The commercial cellulase enzyme was characterized for its optimal pH, temperature, and also studied for the enzyme activity. The effect of organosolvent pretreatment was identified in both native and pretreated wild sugarcane by SEM, XRD, and FTIR. The cellulase enzyme was found to be tolerant with an optimal pH of 5.0 with cellulase activity of pretreated wild sugarcane is found to be 25.36 IU/ml. Organosolvent extraction of wild sugarcane was carried out using water and ethanol as solvent. The composition of lignin, hemicelluloses, and cellulose was also determined in both pretreated and native biomass. The activity of the enzyme was observed over the effect of enzyme loading, substrate loading, temperature, and pH using a Box-Behnken design.

The effect of enzyme loading, alcohol/acid ratio and temperature on the enzymatic esterification of levulinic acid with methanol for methyl levulinate production: a kinetic study.

As an important bio-based chemical, methyl levulinate (ML) can be produced via enzymatic esterification of levulinic acid with methanol. A kinetic model is developed in this work based on the law of mass action and reaction reversibility, to investigate the effect of enzyme loading, alcohol/acid ratio and temperature on ML yield. Data analysis shows that newly developed binary regression is apparently more persuasive than the commonly used unitary regression. Kinetic study reveals: (1) rate constants of esterification/hydrolysis increase with increasing enzyme loading, while their ratio (equilibrium constant) remains invariant. (2) Methanol has no toxicity towards lipase, and hence, neither the rate constants of esterification/hydrolysis nor the equilibrium constant are affected by alcohol/acid ratio. (3) Both rate constants of esterification/hydrolysis and the equilibrium constant increase with temperature elevation, and their relationships agree with Arrhenius equation and Van't Hoff equation, respectively. (4) The esterification is endothermic and spontaneous. In total, the application of binary regression analysis for the developed model to study the enzymatic esterification kinetics is quite successful.

Immobilization of laccase from Trametes hirsuta EDN 082 in light expanded clay aggregate for decolorization of Remazol Brilliant Blue R dye

Immobilization is a method to enhance the activity, the stability and the reusability of enzymes. This study aims to evaluate the capacity of laccase from Trametes hirsuta EDN 082 immobilized in the activated Light Expanded Clay Aggregate (LECA) for dye decolorization. The effects of enzyme loading, the dosage of immobilized laccase-LECA, and the enzyme reusability were investigated. The results showed that the immobilization yield laccase-LECA was 78.78%. One gram of immobilized laccase-LECA with 4.65 U/g of enzyme loading decolorized 99.29% Remazol Brilliant Blue R (RBBR) dye for 3 h. The immobilized laccase-LECA reusability was up to 6 number cycles with 79.67% of the remaining decolorization rate. Immobilized laccase also shifted wide pH and temperature values compare to free enzyme. The kinetic study showed that the specific decolorization rate increases with an increase in initial dye concentration up to 700 and 900 mg/L for laccase-LECA and free laccase, respectively.

Biocatalytic production of isoamyl acetate from fusel oil in supercritical CO2

Isoamyl acetate esters were enzymatically synthesized from fusel oil, a by-product generated in bioethanol industrial plants, in supercritical carbon dioxide (SC-CO2) using immobilized lipase (Lipozyme 435). Effects of enzyme load (2.5–10.0 %), substrates’ molar ratio (acetic anhydride to fusel oil; 1:1–4:1), acyl donor (acetic anhydride, ethyl acetate, and acetic acid) and solvent type (SC-CO2, n-hexane, and solvent-free system) on the biocatalytic isoamyl acetate conversion and the specific productivity were investigated. Higher volumetric expansions of the reaction medium were observed at higher temperature (60 °C) between 10 and 20 MPa. A monophasic system was observed at 15 MPa and 60 °C. Acetic anhydride and SC-CO2 were the best acyl donor and solvent, respectively. An elementary mathematical model was adjusted to the experimental data. 15 MPa, 60 °C, 2:1 substrates’ molar ratio and 2.5 % of enzyme concentration yielded the highest conversion (88.4 ± 0.5 %) and specific productivity (36.9 ± 0.2 %).

Effects of Enzyme Loading and Immobilization Conditions on the Catalytic Features of Lipase From Pseudomonas fluorescens Immobilized on Octyl-Agarose Beads.

The lipase from Pseudomonas fluorescens (PFL) has been immobilized on octyl-agarose beads under 16 different conditions (varying pH, ionic strength, buffer, adding some additives) at two different loadings, 1 and 60 mg of enzyme/g of support with the objective of check if this can alter the biocatalyst features. The activity of the biocatalysts versus p-nitrophenyl butyrate and triacetin and their thermal stability were studied. The different immobilization conditions produced biocatalysts with very different features. Considering the extreme cases, using 1 mg/g preparations, PFL stability changed more than fourfolds, while their activities versus pNPB or triacetin varied a 50–60%. Curiously, PFL specific activity versus triacetin was higher using highly enzyme loaded biocatalysts than using lowly loaded biocatalysts (even by a twofold factor). Moreover, stability of the highly loaded preparations was higher than that of the lowly loaded preparations, in many instances even when using 5°C higher temperatures (e.g., immobilized in the presence of calcium, the highly loaded biocatalysts maintained after 24 h at 75°c a 85% of the initial activity, while the lowly loaded preparation maintained only 27% at 70°C). Using the highly loaded preparations, activity of the different biocatalysts versus pNPB varied almost 1.7-folds and versus triacetin 1.9-folds. In this instance, the changes in stability caused by the immobilization conditions were much more significant, some preparations were almost fully inactivated under conditions where the most stable one maintained more than 80% of the initial activity. Results suggested that immobilization conditions greatly affected the properties of the immobilized PFL, partially by individual molecule different conformation (observed using lowly loaded preparations) but much more relevantly using highly loaded preparations, very likely by altering some enzyme-enzyme intermolecular interactions. There is not an optimal biocatalyst considering all parameters. That way, preparation of biocatalysts using this support may be a powerful tool to tune enzyme features, if carefully controlled.

Enzymatic pre-hydrolysis of organic fraction of municipal solid waste to enhance anaerobic digestion

Enzymatic pretreatment was proposed to enhance the anaerobic digestion of Organic Fraction of Municipal Solid Waste (OFMSW). Enzyme cocktails were produced at laboratory scale from Aspergillus niger fermentation on wheat bran (WB) and OFMSW. The effect of enzyme load, reaction time and agitation speed on OFMSW hydrolysis was determined. Results showed that enzymatic hydrolysis of OFMSW improved organic matter solubilisation. Indeed, pretreatment of OFMSW using WB and OFMSW cocktails resulted respectively in 37.1 and 34.6% change of the soluble chemical oxygen demand and 50 and 40.6% change of the reducing sugars.Anaerobic digestion tests showed that enzyme-treated OFMSW yielded higher biomethane production than raw OFMSW. After hydrolysis by WB and OFMSW cocktails, methane potential of OFMSW increased from 189.2 mL gVS−1 to around 607 and 672 mL gVS−1, respectively. The result confirmed that the use of enzyme cocktails derived from raw waste bioconversion could be a valuable approach to improve biomethane potential of organic wastes.

Immobilization of Thermomyces lanuginosus lipase on multi-walled carbon nanotubes and its application in the hydrolysis of fish oil

In this study, lipase from Thermomyces lanuginosus (TLL) was immobilized on carbon nanotubes (MWCNTs) by physical adsorption, and the immobilizing conditions were optimized for maximum activity. The effects of enzyme loading, pH, temperature and time on the immobilization efficiency and specific activity were evaluated. The highest enzyme activity and immobilization efficiency of 90.66% and 110.5%, respectively, were achieved when the immobilized pH was 8, and a high rate of recovery activity of 111.3% occurred at 45 °C with 30–60 min providing a good result. When the immobilization efficiency was 95.8%, the recovery activity was 112.4%. The immobilization time had little effect on the immobilization efficiency, and 6 mg g−1 of lipase provided the highest immobilization efficiency of 97.78% and recovery activity of 112.8%. When the immobilized lipase was utilized to enrich docosahexaenoic acid (DHA) from fish oil, the DHA content increased with increasing amount of lipase up to 9 mg g−1. The water content had a clear effect when of 50% water was used at 45 °C and at a pH of 7 after 10 h. The DHA contents were 4.2-fold and 2.5-fold greater than the initial content of DHA fish oil for TLL-MWCNTs and free lipase, respectively. The degrees of hydrolysis after 6 cycles of successive use were over 80% and 62% for the immobilized TLL and free TLL, respectively, indicating the system recyclability and the ease of use of the immobilized TLL in industrial applications, especially in the fields of food and medicine.

Highly sensitive, stable and selective hydrogen peroxide amperometric biosensors based on peroxidases from different sources wired by Os-polymer: A comparative study

A comparison was made between two plant peroxidases, cationic horseradish peroxidase (HRP) and anionic tobacco peroxidase (TOP), combined with a highly cationic osmium polymer [Os(4,4′-dimethyl-2,2′-bipyridine)2poly(N-vinylimidazole)10Cl]+2/+ ([Os(dmp)PVI]+/2+) to develop highly sensitive, stable and selective hydrogen peroxide biosensors. The two different plant peroxidases were individually immobilized onto graphite rod (G) electrodes by a three steps drop-casting procedure consisting of the subsequent deposition of an aqueous solution of ([Os(dmp)PVI]+/2+), followed by a solution of poly(ethyleneglycol) diglycidyl ether (PEGDGE), used as a cross linking agent and finally an aliquot of a solution of cationic HRP or anionic TOP to make HRP/PEGDGE/[Os(dmp)PVI]+/2+/G and TOP/PEGDGE/[Os(dmp)PVI]+/2+/G based electrodes, respectively. Electrochemical experiments were carried out to investigate the influence of the surface charge of the enzyme and the charge of the polymer on the efficiency of the electron transfer (ET) between the enzyme and the wiring redox polymer and the efficiency for electrocatalytic reduction of H2O2. In the case of HRP a decrease in the ET rate was observed due to the repulsion between this enzyme and the polymer, both positively charged, whereas with TOP there was an enhanced ET rate due to the attraction between the anionic enzyme and the cationic polymer. The effects of enzyme loading and pH were investigated. Both peroxidase modified electrodes exhibited a wide dynamic response range (1–500μM H2O2) and a low detection limit (0.3μM H2O2). The TOP based electrode showed a higher sensitivity (470nAμM−1cm−2) compared to that of the HRP based electrode (300nAμM−1cm−2) and an improved long-term stability (decrease in 17.3% upon 30days compared with 50% for HRP). Both enzyme electrodes showed a response time of 3s. The HRP based sensor was more sensitive to the presence of phenolic compounds acting as alternative electron donors, whereas the TOP based sensor was virtually interference free. Both HRP and TOP based electrodes were successfully tested in contact lens cleaning samples and real “spiked” samples from different sources such as tap water, milk and dairy products.

Preparation of phytosteryl ester and simultaneous enrichment of stearidonic acid via lipase-catalyzed esterification

Abstract The simultaneous synthesis of a phytosteryl ester and enrichment of stearidonic acid (SDA) were performed via a one-step lipase-catalyzed esterification of fatty acids from Ahiflower™ seed oil with phytosterol. A commercial lipase (Lipase OF) from Candida rugosa was employed as a biocatalyst. Three solvents were screened and cyclohexane was selected as a suitable reaction medium. The effects of enzyme loading, temperature, and solvent amount were investigated. The conversion as well as the SDA content were significantly improved by adding molecular sieves after 1 h of reaction. The optimum conditions were the enzyme loading of 10% (based on the total substrate weight), the temperature of 30 °C, and the solvent amount of 4 mL (based on a substrate weight of 2 g), respectively. The maximum conversion and SDA content in the residual fatty acid were 81 mol% and 58 mol%, respectively, under the optimum conditions. The SDA content increased a 3.4-fold from 17 mol% in Ahiflower™ seed oil to 58 mol%.

Enzymatic Hydrolysis of Steam-Treated Sugarcane Bagasse: Effect of Enzyme Loading and Substrate Total Solids on Its Fractal Kinetic Modeling and Rheological Properties

Rheological studies and fractal kinetic modeling were applied to investigate the enzymatic hydrolysis of steam-exploded sugarcane bagasse (195 °C, 7.5 min) using Cellic CTec3 cellulases (Novozymes). Initially, a central composite rotatable design (CCRD) was performed to evaluate the effect of different enzyme loadings and substrate total solids (TS) on both substrate apparent viscosity and kinetic parameters of enzymatic hydrolysis. Hydrolysis at 20% TS for 12 and 96 h using 38.6 FPU g–1 glucan released 52 and 110 g L–1 glucose equivalents from the steam-exploded material, respectively, with cellobiose being always below 1.7% of these readings. Fractal kinetic modeling provided a good fit of both glucan and xylan conversions, and the fractal kinetic parameters k and h had a strong correlation with changes in both substrate TS and enzyme loading. At the center point of the CCRD, Cellic CTec3 caused a decrease of 1 order of magnitude in the substrate apparent viscosity at every 6 h of hydrolysis. Cellic HTe...

Production of high concentrated cellulosic ethanol by acetone/water oxidized pretreated beech wood

BackgroundLignocellulosic biomass is an abundant and inexpensive resource for biofuel production. Alongside its biotechnological conversion, pretreatment is essential to enable efficient enzymatic hydrolysis by making cellulose susceptible to cellulases. Wet oxidation of biomass, such as acetone/water oxidation, that employs hot acetone, water, and oxygen, has been found to be an attractive pretreatment method for removing lignin while producing less degradation products. The remaining enriched cellulose fraction has the potential to be utilized under high gravity enzymatic saccharification and fermentation processes for the cost-competing production of bioethanol.ResultsBeech wood residual biomass was pretreated following an acetone/water oxidation process aiming at the production of high concentration of cellulosic ethanol. The effect of pressure, reaction time, temperature, and acetone-to-water ratio on the final composition of the pretreated samples was studied for the efficient utilization of the lignocellulosic feedstock. The optimal conditions were acetone/water ratio 1:1, 40 atm initial pressure of 40 vol% O2 gas, and 64 atm at reaction temperature of 175 °C for 2 h incubation. The pretreated beech wood underwent an optimization step studying the effect of enzyme loading and solids content on the enzymatic liquefaction/saccharification prior to fermentation. In a custom designed free-fall mixer at 50 °C for either 6 or 12 h of prehydrolysis using an enzyme loading of 9 mg/g dry matter at 20 wt% initial solids content, high ethanol concentration of 75.9 g/L was obtained.ConclusionThe optimization of the pretreatment process allowed the efficient utilization of beech wood residual biomass for the production of high concentrations of cellulosic ethanol, while obtaining lignin that can be upgraded towards high-added-value chemicals. The threshold of 4 wt% ethanol concentration that is required for the sustainable bioethanol production was surpassed almost twofold, underpinning the efficient conversion of biomass to ethanol and bio-based chemicals on behalf of the biorefinery concept.

Effect of enzyme load and catalyst particle size on the diffusional restrictions in reactions of synthesis and hydrolysis catalyzed by α-chymotrypsin immobilized into glyoxal-agarose

Abstract The magnitude of diffusional restrictions in reactions of peptide hydrolysis and synthesis was studied with α-chymotrypsin immobilized in two different size commercial glyoxal-agarose gel particles at enzyme loads of 0.25, 0.5 and 1 mg protein/g gel. Such magnitude was evaluated by determining the effectiveness factor. Results showed that the effect of diffusional restrictions was stronger for the reaction of hydrolysis than synthesis, being the effectiveness factor in some cases three times higher. Diffusional restrictions were stronger for the catalysts of larger size and with higher enzyme loads, a more than three-fold decrease in the effectiveness factor being observed when the catalyst particle radius increased four times and close to a three-fold decrease when the enzyme load was increased four times. Enzyme loads and particle sizes for avoiding diffusional restrictions in each of the reactions were determined from a steady-state mass balance to the catalyst particle.

Production of Diacylglycerol-enriched Oil by Glycerolysis of Soybean Oil using a Bubble Column Reactor in a Solvent-free System

In this study, diacylglycerol-enriched soybean oil (DESO) was synthesized through Lipozyme 435-catalyzed glycerolysis of soybean oil (SO) in a solvent-free system using a modified bubble column reactor. The effects of enzyme load, mole ratio of glycerol to soybean oil, reaction temperature, gas flow and reaction time on DAG production were investigated. The selected conditions were established as being enzyme load of 4 wt% (mass of substrates), glycerol/soybean oil mole ratio of 20:1, reaction temperature of 80°C, gas flow of 10.6 cm/min, and a reaction time of 2.5 h, obtaining the DAG content of 49.4±0.5 wt%. The reusability of Lipozyme 435 was evaluated by monitoring the contents of DAG, monoacylglycerol (MAG) and triacylglycerol (TAG) in 10 consecutive runs. After purified by one-step molecular distillation, the DAG content of 63.5±0.3 wt% was achieved in DESO. The mole ratio of 1, 3-DAG to 1, 2-DAG was 2:1 and the fatty acid composition had no significant difference from that of soybean oil. However, the thermal properties of DESO and SO had considerable differences. Polymorphic form of DESO were mainly the β form and minor amounts of the β' form. Granular aggregation and round-shaped crystals were detected in DESO.

Synthesis of structured phosphatidylcholine containing punicic acid by the lipase-catalyzed transesterification with pomegranate seed oil

Incorporation of conjugated linolenic acid (punicic acid; 18:3, 9c,11t,13c) into egg-yolk phosphatidylcholine (PC) in the lipase-catalyzed transesterification process was the aim of this work. Pomegranate seed oil (PSO) containing over 77% of punicic acid was used as an acyl donor and three commercially available immobilized lipases were examined as biocatalysts. The effects of enzyme load, reaction time and molar ratio of substrates (PC:PSO) on the incorporation of punicic acid into the modified PC were tested. In all experiments the best results were obtained using lipase B from Candida antarctica. In optimum conditions (20% of enzyme load; 1:3 PC:PSO molar ratio; 72h), the incorporation of punicic acid into the sn-1 position of PC was 56%. Additionally, the total content of polyunsaturated fatty acids in modified PC was almost 50%.

Effects of enzyme loading, densification, and storage on AFEX-pretreated biomass for ethanol production.

Corn stover, switchgrass, and prairie cordgrass were treated with an ammonia fiber expansion (AFEX) process and a novel densification method (ComPAKco). Separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) were used to evaluate impacts of densification. ComPAKco densification is characterized by low-temperature and low-energy requirements, resulting in compacted biomass briquettes (CBB) 1-2cm square, with a bulk density of 380-460kg/m(3). Feedstocks were evaluated before and following AFEX pretreatment, after densification, and after storage. Two enzyme doses were tested. The low rate used 5 filter paper units (FPU) of Spezyme CP (cellulase) and 21.3 cellobiase units (CBU) of Novozyme 188 (aka NS50010 [β-glucosidase]) per gram of glucan. The high dosage rate was three times higher and resulted in 40-56% and 33-82% higher ethanol yields with SHF and SSF, respectively. Trials revealed no adverse effect on ethanol yield following densification or 6-month storage of densified, AFEX-pretreated feedstocks.